Method of making copolymers of amino acids containing glutamic acid



United States Patent 3,076,790 METHOD OF MAKING COPOLYMERS 0F. AMINOACIDS CONTAINING GLUTAMIC ACID Sidney W. Fox, 1114 Waverly Road, andKaoru Harada,

662 W. Call St., both of Tallahassee, Fla. t No Drawing. Filed Aug. 1,1958, Ser. No; 752,403 4 Claims. (Cl. 260-78) Pyroglutamlc acid plusdiketopiperazine plus water b combined glutamic acid and one or moreother neutral and basic a-amino carboxylic acids can be prepared 'byheat-polymerizing glutamic acid or pyroglutamic acid one ormore otherneutral and basic a-amino carboxylic (the 'lactam ofglut arnic acid)together with a balance of acids at-a reaction temperature between about140 and k 210 C., advantageously in the presence of a suificientproportion (preferably equimolar) of concentrated, e.g. about 85 weightpercent or higher, phosphoric acidto facilitate reaction and inthepresence of an inert atmosphere, advantageously carbon dioxide"ornitrogen'; The resultant copolymer is a polypeptide. weight of thecondensation polymersfso jrobtain'edfincreases as the temperature ortime of reaction is increased? In practice, the mixture of amino acids;is heated to temperatures ranging from about 140 to about 210 C.,advantageously in an oil bath, for a time suflicient to give a productof the desired molecular weight, advantageously from about 15 minutes toabout 24 hours.. The

addition of an equimolar amount or less of 85 percent or higherphosphoric acid, amino acid basis, facilitates reaction so that a lowerreaction temperature can be used. The use of phosphoric acid alsopromotes the formation of higher molecular weight polymers and usuallygives higher yields. However, the reaction is operable in the Themolecular 3,076,790 Patented Feb. 5, 1963 min. in the range-of 160-l80C. All of the results are in the following Table I.

Brenna, RESPONSES- or two OR MORE REAOTANTS FOLLOWING HEATING ReactantsBiuret response Glutamic acid plus glycine Glutamic acid plusglycylglyeine f Giutamic acid plus diketopip erazine Iyroglutamic acidplus glycine c Glutainic-Iacid plus asparagine.-. Pyroglutamic acid plusalanine-.. Pyroglutamic acid plus valine.- Pyroglutamic acid plusleucine Pyroglutaniic acid plus phenylalanin Pyroglutauiic acid pluslysine monohydrochloride Pyroglut'amic acid plus leucine plus glycinePyroglutamic acidplus proline plusglycine l Pyiioglutamic acid pluslysine monohydrochloride plus g yr'ma V i Pyi'oglutamic acid plus lysinemonohydrochloride plus ey tine v Pyroglutamic acid plus lysinemonohydrochloride plus glycine plus Oystinp A a On addition of water tothe product, a bluret-positive precipitate separated. g I

b mg. of each reactant, plus iew'drops of water. a A positive biuret isindicative of peptide. d Intensely. V EXAMPLE 2.-'-COPOLYMERIZAT:ION OFGLU- .TAMlC ACID AND GLYCINE A quantity of 1.65 g. (0.01 mole) of'DL-glutamic acid monohydrate was ground with 1.51 g. (0.02 mole) ofglycine in a mortar- The mixture was heated in an open test tube inan'oil bath at 175 -180 C. The mixabsence of addedphosphoric acid,although a starting temperature about 20 C. higher, i.e., C., isrequired in the absence of phosphoric acid. Also, the prior heating ofglutamic acid to. form a melt .of pyroglutamic acid.

EXAMPLE 1.COPOLYMERIZATION GLU- TAMIC ACID AND OTHER AMINO ACIDS 7 blue.

ture melted slowly and evolved a gas which turned litmus When evolutionof gas virtually ceased after 50 filin the heating wasterminatedandthejb'rown liqiii'd solidified on cooling. To this was added 10 ml. ofwater i with rubbing, whereupon a 'white'to" gray solid separated.

After overnight standing, the solids were centrifuged and washed with 10ml. of water and then with 10 ml. of

ethanol. The dried polymer weighed 0.29 g. and gave aniiit'ensebiuretreactioiLas' didthe'rnother liquor. The solid'was-suspended and dialyzedfor several days and then dried in a vacuum dessicator. A gelatin-likefilm was deposited, yield 0.09 g., the average molecular weight of whichwas 16,000. Conducting the, reaction under carbon dioxide orpreferablynitrogen minimized colora' tion which appeared mainly at the surface.

EXAMPLE 3.COPOLYMERIZATION OF PYRO- GLUTAMIC ACID AND GLYCINE Apolymerization was carried out for 4 hr. at

, C. under CO with 44.1 g. (0.03 mole) of L-glutamic In the followingtabulated cases, glutamic acid, L- p or DL- together with another aminoacid in equimolar amount were ground together, then heated, typicallyfor 30120 min. at 160-190 C. The cooled product was treated with 5 or 10ml. of water. In most cases all of the product dissolved. The resultingsolutionwas sub jected to the biuret test with 10 volumes of one-normalNaOH and several drops of 0.3 percem cuso, solution. In other cases, theglutamic acid was first liquefied as the lactam (pyroglutamic acid) byheating, typically for 30 min. at 180 C., and the other component wasthen added in equimolar ratio. Heating was continued usually for acid(preheated for 60 min. at 180 C.) and 56.3 g. (0.75 mole) of glycine.The total yield of slightly colored polymer was 18.2 g. after dialysis.It was difficultly soluble in water and organic solvents, and solubleindilute aqueous sodium bicarbonate solution. Its infrared absorptionmaxima were similarto those of proteins: 3300, 3080, 1690, 1650, 1550,1250 cmr The components recovered after acidic hydrolysis werebiuret-positive and ninhydrin negsuve. i 4' EXAMPLE 4.GLUTAMICACID-GLYCINE COPOLYMERS In thereactions described in the following TableII, the heating was conducted in open test tubes. After heating, thecooled product was treated with 10 ml. of water, left standingovernight, separated by centrifugation, and washed with 10 ml. each ofwater and ethanol. Dialysis was conducted in conventional cellophanetubing for 5 days with agitation of continuously changing water bath bya magnetic stirrer.

Table II YIELDS, TOTAL COMPOSITIONS AND N-TERMINAL COMPOSITIONS OFGLUTAMIC ACID-GLYCINE POLYMER Yield of polymer Total compositionN-Terminal Moles and Time of form of Moles 01' Temp., heating, GlutarnicN-glutamic N-Glycine/ glutamic glycine 0. min. Before After acid,Glycine, acid/EN- EN-amlno acid dialysis, dialysis, mol. mol. ammo acid,acid, mol.

5. g. percent percent mol. percent percent 0.01 DL 0.02 175-180 50 0.290.09 24 76 0.01 L 0. 02 175-180 50 0.29 0. 09 23. 4 76. 6 0.01 L 0.03175-180 60 0. 55 0. 28 20. 2 79. 8 17 83 Not determined. e Pyroglutamicacid from L-glutamic acid.

EXAMPLE 7.-EFFECT OF PHOSPI-IORIC ACID ON INFRARED ABSORPTION MAXIMA OF20 REACTION BETWEEN 0.01 MOLE ASPARTIC POLY (GLUTAMIC ACID GLYCINE) ACIDAND 0.01 MOLE GLUTAMIC ACID, ONE

HOUR REACTION TIME Absorption Interpretation band, Cm Yields afterdialysis, g.

p 'lemp., C. a, Nn stmtchin g Without With Avg. mol. 3, 08 0 HgPO; 113104 weight 1,690 00 of coon. 1, 650 C0 stretching, amide I.

1, 550 NH deformation, amide II. 0 0 44 1, 250 Amide III. Trace 11 e1IIIIIIIIIIII 0. 89 1. 76 6, 740 1. 21 2. 09 11, 800 1 On basis of 23.4glutamic acid: 76.6 glycine, as reported in Table II. 1. 95 2. is 251600Nora-The spectra show that the material has a typical linear polypeptidestructure, and is not a diketopiperazine.

N OTE.-A11 equimolar proportion of aqueous weight percent 11; P0

was used, amino acid basis.

EXAMPLE 5.COPOLYMERIZATION OF GLUTAMIC ACID AND GLYCINB [Temperature vs.mol. wt.]

Yield of polymer N-terminal Nondifi'usible Glycine Temp., polymer (A)!Glutamic acid content Ave., mol.

C. total polymer, content of A, of A, mol. weight of A N-glutamlcN-glycine/ Before 11 After weight percent mol. percent percent acid/Z N-Z N-amino dialysis, g. dialysis, g. amino acid, acid, mol.

mol. percent percent O. 00 Trace 0.60 d 0.37 0 17 24. l 75. 9 15, 500 2377 0.68 e 0. 47 69 2 25. 5 74. 5 17, 500 21 79 0.70 0. 57 81 4 25. 7 74.3 20, 000 19 81 L-glutamic acid (0.01 mole) was heated at 180C. for 30min., treated with 0.025 mole of glycine in an open tube with heatingfor 60 min, in an oil bath at the temperature given.

b The reaction mixture was taken up in 10 ml. of Water, let standovernight, separated at the centrifuge and washed with 10 ml. of waterand 10 m1. of ethanol.

0 A iter drying in vacuum desiccator following 7 days of dialysis.

6 Yellow film, useful as water-removable protective film at lowhumidities.

Q Grey film, useful as water-removable protective film at lowhumidities.

I Grey-black film, useful as water-removabie protective film at lowhumidities.

EXAMPLE 6.COPOLYMERIZATION OF GLUTAMIC ACID AND GLYCINE Yield of polymerN-Terminal Nondifiusible Glutamic Glycine Reaction overlaying polymer(A)/ acid content, time hr. atmosphere Before After total polymercontent, mol. N-giutamie N-glycine/ dialysis, dialysis, Weight mol.percent acid/E N- 2 N-arnino g. {5. percent percent amino acid, acid,mol.

mol. percent percent 0.25 0. 12 48 22. 6 77. 4 O. 52 0. 39 75 27. 1 72.9 2O 80" 0. 62 0. 51 82 26. 7 73. 3 19 81 0. 80 0. 71 80 33. 2 66. 8 1783 0.26 0. 04 15 O. 43 0. 15 35 0.63 0 32 51 A All mixtures contained0.01 mole of L-glutamic acid which was heated at 180C. then treated with0.025 mole oi glycine at 170 C. for the time given, then processed as inExample 5.

b Almost colorless film.

" Slightly grey film.

I Dark grey film.

1 Not determined:

AMINO ACID POLYMER COMPOSITION DETERMINATION Polymer (100 mg.) ishydrolyzed with ml. of 6- normal hydrochloric acid under reflux for 6hours, and the dinitrophenylamino acid derivatives are prepared as inI.A.C.S. 76; 1328 (1954), as follows. After concentration under reducedpressure, water is added and the process is repeated to remove unboundhydrochloric acid. The residue is neutralized to pH 7 with aqueoussodium bicarbonate solution. To this mixture, which usually is of about5 ml. volume, is added 0.4 g. of sodium bicarbonate, 0.4 g. (2.2millimoles, 0.28 ml.) of 2,4-dinitrofiuorobenzene and 10 ml. of ethanol.After 2 hr. of mechanical shaking and 12 hr. of standing in the dark,the ethanol is evaporated under reduced pressure at C. to approximatelyone-fourth the original volume. The liquid is acidified with 2-normalhydrochloric acid and extracted thrice with ethyl acetate. After theethyl acetate is evaporated, 10 ml. of chloroform-ether (2:1) is addedand the resulting dinitrophenyl-amino acid derivatives and dinitrophenolare separated from one another by chromatography on Hyfio Superceldiatomaceous silica, as described in Nature 167: 513 (1951). Theabsorbent is prepared by treating it with 0.2 molar buffer (sodiumdihydrogen phosphate and citric acid, pH 4) and packing it solidly intoa tube of 8 mm. internal diameter and 25 cm. length. The column ischarged with the chloroformether solution and the individual bands ofdinitrophenylglycine and the dinitrophenylglutamic acid compounds areseparated, and the solvent is evaporated. Each fraction is eluted with1.5 percent sodium bicarbonate solution and centrifuged to separatetraces of silica carried along. The optical density of each of thesesolutions is read on a Beckman spectrophotometer at 360 mg and comparedwith a standard solution of the dinitrophenylamino acid compound inorder to estimate the amount. ESTIMATION OF PROPORTIONS OF N-GLUTAMICACID AND N-GLYCINE Dinitrophenyl derivative of amino acid polymer to 100mg.) is hydrolyzed with 25 ml. of 6-norma1 hydrochloric acid underreflux for 12 hrs. in the dark. The solution is diluted with an equalvolume of Water, extracted by ethyl acetate, the ethyl acetate'isevaporated, and the residue is dissolved in chloroform-ether. Thedinitrophenyl-amino acid derivatives are separated by chromatography andestimated as described earlier, with the superposition of correctionscalculated from recovery experiments with dinitrophenylglutamie acid anddinitrophenylglycine polymer-s hydrolyzed under the same con- 6 ditionsas dinitrophenyl-polymer; F. Sanger, Biochem. J. 39, 507 (1945).ESTIMATION OF AVERAGE MOLECULAR WEIGHT 0F PEPTIDE CHAIN The analyticalprocedure is the same as for assay of N-amino acids; W. R. Middlebrook,Biochim. Biophys. Acta 7: 547 (1951). The molecular weights arecalculated on the basis of a polymer consisting of 25 mole percentglutamic acid residue and percent glycine residue. This calculationassumes no fragmentation of polymer molecule during thedinitrophenylation, no cyclopeptide, and no N-terminal pyroglutamicacid.

In order to check on whether the amino group assessed by thedinitrofiuorobenzene might arise from a pyroglutamyl residue opened bythe dinitrophenylation, the dinitrophenylation procedure was carried outon an authentic sample of DL-pyroglutamic acid. No colored product otherthan dinitrophenol was obtained.

What is claimed is:

1. Method for making a polypeptide by heating a mixture which consistsof glutamic acid and at least one other tit-amino carboxylic acid of thegroup consisting of neutral and basic amino acids at temperaturesranging between about 160 and about 210 C. until a polypeptide hasformed.

2. Method of claim 1, wherein the glutamic acid is heat-polymerized withglycine.

3. Method of claim 1, wherein the glutamic acid is first heated to forma melt of pyroglutamic acid.

4. Method of claim 1, wherein a substantially equimolar amount, aminoacid basis, of aqueous percent phosphoric acid is added and the reactiontemperature ranges between about and 210 C.

References Cited in the file of this patent UNITED STATES PATENTSCarothers Feb. 16, 1937 Hanford Aug. 18, 1942 OTHER REFERENCES

1. METHOD FOR MAKING A POLYPEPTIDE BY HEATING A MIXTURE WHICH CONSISTSOF GLUTAMIC ACID AND AT LEAST ONE OTHER A-AMINO CARBOXYLIC ACID OF THEGROUP CONSISTING OF NEUTRAL AND BASIC AMINO ACIDS AT TEMPERATURESRANGING BETWEEN ABOUT 160* AND ABOUT 210*C. UNTIL A POLYPEPTIDE HASFORMED.